TNFSF Single Chain Molecules

ABSTRACT

The present invention refers to single-chain fusion proteins comprising three soluble TNF superfamily (TNFSF) cytokine domains and nucleic acid molecules encoding these fusion proteins. The fusion proteins are substantially non-aggregating and suitable for therapeutic, diagnostic and/or research applications.

The present invention refers to single-chain fusion proteins comprisingthree soluble TNF superfamily (TNFSF) cytokine domains and nucleic acidmolecules encoding the fusion proteins. The fusion proteins aresubstantially non-aggregating and suitable for therapeutic, diagnosticand/or research applications.

STATE OF THE ART

It is known that trimerisation of TNFSF cytokines, e.g., the CD95 ligand(CD95L), is required for efficient receptor binding and activation.Trimeric complexes of TNF superfamily cytokines, however, are difficultto prepare from recombinant monomeric units.

WO 01/49866 and WO 02/09055 disclose recombinant fusion proteinscomprising a TNF cytokine and a multimerisation component, particularlya protein from the C1q protein family or a collectin. A disadvantage ofthese fusion proteins is, however, that the trimerisation domain usuallyhas a large molecular weight and/or that the trimerisation is ratherinefficient.

Schneider et al. (J Exp Med 187 (1989), 1205-1213) describe that trimersof TNF cytokines are stabilised by N-terminally positioned stabilisationmotifs. In CD95L, the stabilisation of the receptor binding domaintrimer is presumably caused by N-terminal amino acid domains which arelocated near the cytoplasmic membrane.

Shiraishi et al. (Biochem Biophys Res Commun 322 (2004), 197-202)describe that the receptor binding domain of CD95L may be stabilised byN-terminally positioned artificial α-helical coiled-coil (leucinezipper) motifs. It was found, however, that the orientation of thepolypeptide chains to each other, e.g. parallel or antiparallelorientation, can hardly be predicted. Further, the optimal number ofheptad-repeats in the coiled-coil zipper motif are difficult todetermine. In addition, coiled-coil structures have the tendency to formmacromolecular aggregates after alteration of pH and/or ionic strength.

WO 01/25277 relates to single-chain oligomeric polypeptides which bindto an extracellular ligand binding domain of a cellular receptor,wherein the polypeptide comprises at least three receptor binding sitesof which at least one is capable of binding to a ligand binding domainof the cellular receptor and at least one is incapable of effectivelybinding to a ligand binding domain of the cellular receptor, whereby thesingle-chain oligomeric polypeptides are capable of binding to thereceptor, but incapable of activating the receptor. For example, themonomers are derived from cytokine ligands of the TNF family,particularly from TNF-α.

WO 2005/103077 discloses single-chain fusion polypeptides comprising atleast three monomers of a TNF family ligand member and at least twopeptide linkers that link the monomers of the TNF ligand family membersto one another. Recent experiments, however, have shown that thesesingle-chain fusion polypeptides show undesired aggregation.

It was an object of the present invention to provide single-chain fusionproteins comprising at least three TNF cytokine domains which allowefficient recombinant manufacturing combined with good stabilityconcerning aggregation.

SUMMARY OF THE INVENTION

The present invention relates to a single-chain fusion polypeptidecomprising:

-   -   (i) a first soluble TNF-family cytokine domain,    -   (ii) a first peptide linker,    -   (iii) a second soluble TNF-family cytokine domain,    -   (iv) a second peptide linker, and    -   (v) a third soluble TNF-family cytokine domain,        which is substantially non-aggregating.

The invention further relates to a nucleic acid molecule encoding afusion protein as described herein and to a cell or a non-human organismtransformed or transfected with a nucleic acid molecule as describedherein.

The invention also relates to a pharmaceutical or diagnostic compositioncomprising as an active agent a fusion protein, a nucleic acid molecule,or a cell as described herein.

The invention also relates to a fusion protein, a nucleic acid molecule,or a cell as described herein for use in therapy, e.g., the use of afusion protein, a nucleic acid molecule, or a cell as described hereinfor the preparation of a pharmaceutical composition in the prophylaxisand/or treatment of disorders caused by, associated with and/oraccompanied by dysfunction of TNFSF cytokines, particularlyproliferative disorders, such as tumours, e.g. solid or lymphatictumours; infectious diseases; inflammatory diseases; metabolic diseases;autoimmune disorders, e.g. rheumatoid and/or arthritic diseases;degenerative diseases, e.g. neurodegenerative diseases such as multiplesclerosis; apoptosis-associated diseases or transplant rejections.

DESCRIPTION OF THE FIGURES

FIG. 1 Domain structure of the inventive single-chain fusionpolypeptide. I., II., III. soluble TNF-family cytokine domains.

FIG. 2 Schematic picture representing the general structure of TNF-SFproteins. ▪▪▪ cell membrane, N-terminus located within the cell, 1.anti-parallel β-fold of receptor-binding domain (RBD), 2. interface ofRBD and cell membrane, 3. protease cleavage site.

FIG. 3 Schematic picture representing the structure of the native TNF-SFtrimer. Cylindric structures represent RBDs, N-termini connect RBD withthe cell membrane.

FIG. 4 Schematic picture representing the structure of three solubledomains comprising the receptor-binding domain of a TNF cytokine. I.,II., III. soluble TNF-family cytokine domains.

FIG. 5 Trimerisation of the soluble domains comprising the RBD of a TNFcytokine, characterised in that the N- and C-termini of the threesoluble domains form a surface.

FIG. 6 Schematic picture representing the structure of the single-chainTNF-SF comprising all or a part of the stalk-region illustrating therequirement of longer linkers to compensate for the distance to theN-terminus of the next soluble domain.

FIG. 7 scFv-TNF-SF fusion protein known from the art.

FIG. 8 Fc-TNF-SF fusion protein known from the art.

FIG. 9 9A Single-chain fusion polypeptide comprising an additional Fabantibody fragment. 9B Single-chain fusion polypeptide comprising anadditional scFv antibody fragment.

FIG. 10 Dimerisation of two N-terminally fused scFc fusion polypeptidesvia disulfide bridges.

FIG. 11 Dimerisation of two C-terminally fused scFc fusion polypeptidesvia disulfide bridges.

FIG. 12 Dimerisation of single-chain fusion polypeptides via a linker.

FIG. 13 Single-chain fusion polypeptide comprising an additional Fabantibody fragment further fused to a second fusion polypeptide or to ascFv fusion polypeptide.

FIG. 14 Dimerisation of two scFab fusion polypeptides via disulfidebridges.

FIG. 15 N-terminally fused scFc fusion polypeptides further comprising aFv and/or Fab antibody fragment.

FIG. 16 C-terminally fused scFc fusion polypeptides further comprising aFv and/or Fab antibody fragment.

FIG. 17 SEC analysis of recombinantly expressed, purified TNF-SF membersunder native conditions. Exemplarily shown are two SEC analyses ofpurified TNF-SF members on a Superdex200 column under native condition(e.g.: PBS, pH 7.4). The diagrams show the absorption at 280 nm (mAU)plotted against the elution volume (ml). The filled arrow indicates theelution peak for the fraction containing defined, soluble trimericTNF-SF protein. The triangle indicates the elution peak for theoligomerised TNF-SF The open arrow indicates the void volume of theSEC-column that contains protein-aggregates, which are too big to beseparated (>800 kDa).

FIG. 17 A: TNF-SF protein Aggregation Diagram A exemplarily shows ananalysis of a TNF-SF protein preparation that contains a high amount ofoligomerised/aggregated protein (indicated by the high amount of proteineluting in the void volume and the high amount of oligomeric protein).

FIG. 17 B: TNF-SF protein defined soluble protein Diagram B exemplarilyshows an analysis for a TNF-SF protein preparation that contains almostexclusively defined soluble protein (indicated by the absence of proteineluting in the void volume and by the very limited amount of proteineluting as oligomer).

FIG. 18 SEC analysis of recombinantly expressed, affinity purifiedFab-scTRAILR2-SSSS,

-   -   SEC analysis of Fab-scTRAILR2-SSSS on a Superdex200 column using        PBS, pH 7.4. The diagram shows the absorption at 280 nm (mAU)        plotted against the elution volume (ml). The protein elutes as a        distinct peak with an elution volume of 14.56 ml, corresponding        to an apparent MW of 68 kDa. No additional protein peaks with        lower retention volume, indicating oligomerised/aggregated        protein, could be observed.

FIG. 19 SEC analysis of recombinantly expressed, affinity purifiedFab-scTRAILR2-SNSN.

-   -   SEC analysis of Fab-scTRAILR2-SNSN on a Superdex200 column using        PBS, pH 7.4. The diagram shows the absorption at 280 nm (mAU)        plotted against the elution volume (ml). The protein elutes as a        distinct peak with an elution volume of 14.12 ml, corresponding        to an apparent MW of 87 kDa. No additional protein peaks with        lower retention volume, indicating oligomerised/aggregated        protein, could be observed.

FIG. 20 SEC analysis of recombinantly expressed, affinity purifiedFab-scTRAILwt-SNSN.

-   -   SEC analysis of Fab-scTRAILwt-SNSN on a Superdex200 column using        PBS, pH 7.4. The diagram shows the absorption at 280 nm (mAU)        plotted against the elution volume (ml). The protein elutes as a        distinct peak with an elution volume of 13.99 ml, corresponding        to an apparent MW of 94 kDa. A small additional protein peak at        12.00 ml could be observed. The apparent Mw of this peak        corresponds to about 270 kDa, indicating a defined trimerisation        of Fab-scTRAILwt-SNSN. The total protein amount of the peak at        12.00 ml accounts for <3% of the total protein. More than 97% of        the analysed Fab-scTRAILwt-SNSN has a defined soluble state        (correct assembly of the three receptor binding modules). The        peak at 16.12 ml corresponding to a MW of 28 kDa contains        Fab-light-chain polypeptide and was not included for the        analysis of peak areas.

FIG. 21 Human scTRAIL Linker glycosylation

FIG. 21A Amino acid sequence of the linker(s) used to combine thereceptor binding modules of single chain TRAIL constructs. Gly281encodes the last amino acid of a respective receptor binding module, thesequence GSGN/SGN/SGS encodes the linker sequence, Arg121 encodes thefirst amino acid of the following TRAIL receptor binding domain. Thedesigned linker sequences contains two putative N-linked glycosylationsites at position 1 or 2 as indicated. These positions were permutatedas indicated (version I, II, III).

FIG. 21B Combination of linker positions: The scTRAIL molecules containthree homologue modules (grey barrels) that are connected with linker 1and linker 2 as indicated. Each of the two linkers, can be designed forN-linked glycosylation as described in “A”. A complete set of 9different proteins containing all possible combinations of linkers canbe designed based on the sequences shown in B for linker 1 and 2. (Sixof these proteins were expressed—see “C”).

FIG. 21C Nomenclature of scTRAIL constructs expressed to test theinfluence of different linker sequences on glycosylation

FIG. 22 Western Blot analysis of recombinant scTRAIL constructs

-   -   Single chain TRAIL proteins with different linker sequences were        recombinantly expressed, separated by SDS-PAGE and transferred        to a PVDF-membrane. Bound proteins were detected with a mouse        monoclonal antibody recognising the Strep-Tag followed by a        Peroxidase-conjugated secondary anti-mouse antibody. Different        TRAIL variants were loaded as indicated. Note the MW-shift        indicating differential glycosylation of scTRAIL-linker        variants.

FIG. 23 Cell culture supernatant of HEK293 cells, transiently expressingscCD95L (SEQ-ID NO:27) was collected and used to stimulate Jurkat cellsat varying concentrations. The supernatant was used either directlywithout further modifications or an anti-Streptag antibody (2microgram/ml) was added to cross-link the scCD95L protein. Jurkat cellswere incubated with HEK293 cell culture supernatant for three hours at37°, lysed and analysed for caspase activity. Only cell supernatant thatcontained cross-linked scCD95L-St increased caspase activity in Jurkatcells, indicating that scCD95L alone does not form higher orderaggregates able to be pro-apoptotic.

FIG. 24 The protein scCD95L (SEQ ID NO:27) can be produced by transienttransfection of HEK293 cells, stable transfection of other eukaryoticcells or by expression using prokaryotic cells. The recombinant proteincan be affinity purified by using StrepTactin Sepharose matrix. Boundprotein can be eluted with a buffer containing desthio-biotin. FIG. 2shows a silver stained SDS-PAGE of the elution fractions (lanes 1 to 5;fraction 2 is positive) of the affinity purification. The elutionfraction containing scCD95L could be applied to size exclusionchromatography (SEC). It is expected, that the protein shows only a lowaggregate content.

FIG. 25 Cell culture supernatants of HEK293 cells, transientlyexpressing single chain TRAIL proteins with different linkers (derivedfrom SEQ ID 28) were collected and used to stimulate Jurkat cells atvarying dilutions (exemplarily, a dilution of 1:8 is shown in thisfigure). The supernatants were used either directly without furthermodifications or an anti-Streptag antibody (2 microgram/ml Strep MABImmo) was added to cross-link the scTRAIL proteins. Jurkat cells wereincubated with HEK293 cell culture supernatant for three hours at 37°,lysed and analysed for caspase activity. Cell culture supernatant thatcontained cross-linked scTRAILwt proteins induced an increased caspaseactivity in Jurkat cells, indicating that scTRAILwt proteins alone doform only a low amount of higher order aggregates able to bepro-apoptotic.

FIG. 26 Influence of the module succession of scTRAIL-constructcomponents on their expression rate of Fab-scTRAIL fusion proteins.Western blot of HEK293T cell culture supernatants from transientexpression experiments. The polypeptide chains necessary for theformation of the Fab-scTRAIL proteins were either expressed separately(lanes 1 to 10) or alternatively co-expression experiments wereperformed (lanes 11-13). After reducing SDS-PAGE, proteins weretransferred to a nitrocellulose membrane and proteins containing aStreptag werde detected, using an anti-Streptag specific mAB as primaryAB. The light-chain-scTRAIL(R2-specific) proteins were secreted even inthe absence of the accessory heavy chain (lanes 1-4). In contrast, theheavy-chain-scTRAIL(R2-specific) fusion proteins were not secreted inthe absence of the accessory light chain (lanes 5-8). As exemplified inlane 13., the heavy-chain-scTRAIL(R2-specific) fusion proteins were onlysecreted in the presence of the light chain.

FIG. 27 Cell culture supernatants of HEK293T cells, transientlyexpressing scTRAILwt-Fc fusion proteins with different linkers werecollected and used to stimulate Jurkat cells at varying dilutions. Thesupernatants were used directly without further modifications (FigureXX-A). Jurkat cells were incubated with HEK293T cell culture supernatantfor three hours at 37°, lysed and analysed for caspase activity. Therewas already a pronounced proapoptotic capacity present in thescTRAILwt-Fc containing supernatants, indicating that scTRAILwt-Fcfusion proteins alone do form dimeric assemblies able to bepro-apoptotic.

FIG. 28 It is well known that the use of artificially cross-linked or amembrane-bound ligand of the TNF superfamily has superior bioactivity ascompared to soluble, homotrimeric ligand. Thus the local enrichment ofsingle chain TRAIL (scTRAIL) constructs on cells that express theantigen Her2 via the Her2-selective Fab-fragment (“Pertuzumab”) fused tothese scTRAIL proteins should increase their cytotoxic bioactivity.Likewise, the blocking of the Her2 binding sites on cells bypre-incubation with the Her2-specific Fab-fragment (Pertuzumab-Fab) onlyshould decrease the cytotoxic bioactivity of Fab-scTRAIL fusionproteins. As shown in FIG. 28A, scTRAIL constructs induce the death ofHT1080 cells, as the viability decreases with increasing proteinconcentration. In accordance, the pre-incubation of HT1080 cells withthe Fab-fragment (Pertuzumab-Fab), followed by co-incubation with theFab-scTRAIL constructs (Fab-scTRAILR2-SNSN or Fab-scTRAILwt-SNSN) overnight, reduced the cytotoxic activity of the Fab-scTRAIL constructs(FIG. 28B), whereas the Fab only induced no cell death (Pertuzumab-Fab).This means that the Fab-scTRAIL constructs bind to HT1080 cells via theFab fragment thus increasing the cytotoxic bioactivity of scTRAIL.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention a substantially non-aggregatingfusion polypeptide comprising at least three soluble TNF family liganddomains connected by two peptide linkers is provided.

The term “non-aggregating” refers to a monomer content of thepreparation of ≧50%, preferably ≧70% and more preferably ≧90%. The ratioof monomer content to aggregate content may be determined by examiningthe amount of aggregate formation using size-exclusion chromatography(SEC). The stability concerning aggregation may be determined by SECafter defined time periods, e.g. from a few to several days, to weeksand months under different storage conditions, e.g. at 4° C. or 25° C.For the fusion protein, in order to be classified as substantiallynon-aggregating, it is preferred that the monomer content is as definedabove after a time period of several days, e.g. 10 days, more preferablyafter several weeks, e.g. 2, 3 or 4 weeks, and most preferably afterseveral months, e.g. 2 or 3 months of storage at 4° C., or 25° C.

As an increase of e.g. the apoptosis inducing potential in the case ofscCD95L on human Jurkat cells correlates with its aggregation state, thestability of the fusion polypeptide concerning aggregation may also bedetermined by examining the biological activity of the fusionpolypeptide.

The single-chain fusion polypeptide may comprise additional domainswhich may be located at the N- and/or C-termini thereof. Examples foradditional fusion domains are e.g. single-chain antibodies or antibodyfragments or other targeting molecules or a further cytokine domain,e.g. an interleukin.

The single-chain fusion protein comprises three soluble domains derivedfrom a cytokine of the TNF superfamily. Preferably, those solubledomains are derived from a mammalian, particularly human cytokineincluding allelic variants and/or derivatives thereof. The solubledomains comprise the extracellular portion of a TNFSF cytokine includingthe receptor binding domain without membrane located domains. Proteinsof the TNF superfamily are anchored to the membrane via an N-terminalportion of 15-30 amino acids, the so-called stalk-region. The stalkregion contributes to trimerisation and provides a certain distance tothe cell membrane. However, the stalk region is not part of the receptorbinding domain (RBD).

Importantly, the RBD is characterised by a particular localisation ofits N- and C-terminal amino acids. Said amino acids are immediatelyadjacent and are located centrally to the axis of the trimer. The firstN-terminal amino acids of the RBD form an anti-parallel beta-strand withthe C-terminal amino acids of the RBD (FIGS. 2 and 3).

Thus, the anti-parallel beta-strand of the RBD forms an interface withthe cell membrane, which is connected to and anchored within the cellmembrane via the amino acids of the stalk region. It is highly preferredthat the soluble domains of the single-chain fusion protein comprises areceptor binding domain of the TNF-SF cytokine lacking any amino acidsfrom the stalk region (FIGS. 4 and 5). Otherwise, a long linkerconnecting the C-terminus of one of the soluble domains with theN-terminus of the next soluble domain would be required to compensatefor the N-terminal stalk-region of the next soluble domain (FIG. 6),which might result in instability and/or formation of aggregates.

A further advantage of such soluble domains is that the N- andC-terminal amino acids of the RBD are not accessible for any anti-drugantibodies.

Preferably, the single-chain fusion polypeptide is capable of forming anordered trimeric structure comprising at least one functional bindingsite for the respective cytokine receptor.

The fusion polypeptide may comprise one, two or three functionalcytokine receptor binding sites, i.e. amino acid sequences capable offorming a complex with a cytokine receptor. Thus, at least one of thesoluble domains is capable of binding to the corresponding cytokinereceptor. In one embodiment, at least one of the soluble domains iscapable of receptor activation, whereby apoptotic and/or proliferativeactivity may be effected. In a further embodiment, one or more of thesoluble domains are selected as not being capable of receptoractivation.

The soluble domain may be derived from TNF superfamily members, e.g.human TNFSF-1 to -18 and EDA-A1 to -A2 as indicated in Table 1,preferably from LTA (SEQ ID NO:1), TNFα (SEQ ID NO:2), LTB (SEQ IDNO:3), OX40L (SEQ ID NO:4), CD40L (SEQ ID NO:5), CD95L (SEQ ID NO:6),CD27L (SEQ ID NO:7), CD30L (SEQ ID NO:8), CD137L (SEQ ID NO:9), TRAIL(SEQ ID NO:10), RANKL (SEQ ID NO:11), TWEAK (SEQ ID NO:12), APRIL 1 (SEQID NO:13), APRIL 2 (SEQ ID NO:14), BAFF (SEQ ID NO:15), LIGHT (SEQ IDNO:16), TL1A (SEQ ID NO:17), GITRL (SEQ ID NO:18), EDA-A1 (SEQ ID NO:19)and EDA-A2 (SEQ ID NO:20). Preferred soluble domains of the respectiveproteins are indicated in Table 1 (NH₂-aa to COOH-aa) and, e.g.,comprise amino acids 59-205, 60-205 or 64-205 of LTA (SEQ ID NO:1),86-233 of TNFα (SEQ ID NO:2), 82-244 or 86-244 of LTB (SEQ ID NO:3),52-183 or 55-183 of OX40L (SEQ ID NO:4), 112-261, 117-261 or 121-261 ofCD40L (SEQ ID NO:5), 51-193 or 56-193 of CD27L (SEQ ID NO:7), 97-234,98-234 or 102-234 of CD30L (SEQ ID NO:8), 86-254 of CD137L (SEQ IDNO:9), 161-317 of RANKL (SEQ ID NO:11), 103-249, 104-249, 105-249 or106-249 of TWEAK (SEQ ID NO:12), 112-247 of APRIL 1 (SEQ ID NO:13),112-250 of APRIL 2 (SEQ ID NO:14), 140-285 of BAFF (SEQ ID NO:15),91-251, 93-251 or 97-251 of TL1A (SEQ ID NO:17), 52-177 of GITRL (SEQ IDNO:18), 245-391 of EDA-A1 (SEQ ID NO:19), 245-389 of EDA-A2 (SEQ IDNO:20). More preferably, the soluble domains are derived from CD95L,TRAIL or LIGHT. In an especially preferred embodiment, the solubledomains are selected from human CD95L, particularly starting from aminoacids 144, 145 or 146 and comprise particularly amino acids 144-281 or145-281 or 146-281 of SEQ ID NO:6 or human TRAIL, particularly startingfrom amino acids 120-122 and comprise particularly amino acids 120-281,121-281 or 122-281 of SEQ ID NO:10. Optionally, amino acid Lys145 of SEQID NO:6 may be replaced by a non-charged amino acid, e.g. Ser or Gly.Optionally, amino acid Arg121 of SEQ ID NO:10 may be replaced by anon-charged amino acid, e.g. Ser or Gly. In a further preferredembodiment, the soluble domains are selected from human LIGHT,particularly starting from amino acids 93, 94 or 95 of SEQ ID NO:16 andparticularly comprise amino acids 93-240, 94-240 or 95-240 of SEQ IDNO:16.

As indicated above, the soluble domains may comprise the wild-typesequences as indicated in SEQ ID NO: 1-20. It should be noted, however,that it is possible to introduce mutations in one or more of thesesoluble domains, e.g. mutations which alter (e.g. increase or decrease)the binding properties of the soluble domains. In one embodiment,soluble domains may be selected which cannnot bind to the correspondingcytokine receptor. An example of such a mutation is a replacement ofamino acid Y218 in human CD95L (SEQ ID NO:6) by another amino acid, e.g.R, K, S or D. Further, a mutation may be introduced which alters thebinding to other cellular and/or extracellular components, e.g. theextracellular matrix. An example of such a mutation is a replacement ofamino acid K177 in CD95L (SEQ ID NO: 6) by another amino acid, e.g. E, Dor S.

In a further preferred embodiment of the invention, the soluble cytokinedomain (i) comprises a mutant of the cytokine of the TNF superfamily ora receptor binding domain thereof which binds and/or activatesTRAIL-receptor 1 (TRAILR1) and/or TRAIL-receptor 2 (TRAILR2). Thebinding and/or activity of the mutant may be, e.g., determined by theassays as described in van der Sloot et al, (PNAS, 2006, 103:8634-8639),Kelley et al. (J. Biol. Chem., 2005, 280:2205-2215), or MacFarlane etal. (Cancer Res., 2005, 65: 11265-11270).

The mutant may be generated by any technique and is known by the skilledperson, e.g., the techniques described in van der Sloot et al. (PNAS,2006, 103:8634-8639), Kelley et al. (J. Biol. Chem., 2005,280:2205-2215), or MacFarlane et al. (Cancer Res., 2005, 65:11265-11270) and may comprise any type of structural mutations, e.g.,substitution, deletion, duplication and/or insertion of an amino acid. Apreferred embodiment is the generation of substitutions. Thesubstitution may affect at least one amino acid of the cytokine of theTNF superfamily or a receptor binding domain thereof as describedherein. In a preferred embodiment, the substitution may affect at leastone of the amino acids of TRAIL, e.g., human TRAIL (e.g., SEQ ID NO:10). Preferred substitutions in this regard affect at least one of thefollowing amino acids of human TRAIL of SEQ ID NO:10: R130, G160, Y189,R191, Q193, E195, N199, K201, Y213, T214, S215, H264, I266, D267, D269.Preferred amino acid substitutions of human TRAIL of SEQ ID NO:10 are atleast one of the following substitutions: R130E, G160M, Y189A, Y189Q,R191K, Q193S, Q193R, E195R, N199V, N199R, K201R, Y213W, T214R, S215D,H264R, I266L, D267Q, D269H, D269R, or D269K.

The amino acid substitution(s) may affect the binding and/or activity ofTRAIL, e.g., human TRAIL, to or on either the TRAILR1 or the TRAILR2.Alternatively, the amino acid substitution(s) may affect the bindingand/or activity of TRAIL, e.g., human TRAIL, to or on both, the TRAILR1and the TRAILR2. The binding and/or activity of the TRAILR1 and/orTRAILR2 may be affected positively, i.e., stronger, more selective ormore specific binding and/or more activation of the receptor.Alternatively, the binding and/or activity of the TRAILR1 and/or TRAILR2may be affected negatively, i.e., weaker, less selective or lessspecific binding and/or less or no activation of the receptor.

Examples of mutants of TRAIL with amino acid substitution(s) of theinvention that affect binding and/or activation of both TRAILR1 andTRAILR2 may be found, e.g., in Table 1 of MacFarlane et al. (cf, above)and may comprise a human TRAIL mutant with the following two amino acidsubstitutions of SEQ ID NO: 10 Y213W and S215D or with the followingsingle amino acid substitution: Y189A.

Examples of mutants of TRAIL with amino acid substitution(s) of theinvention that affect binding and/or activation of TRAILR1 may be found,e.g., in Table 1 of MacFarlane et al. (cf. above) and may comprise ahuman TRAIL mutant with the following four amino acid substitutions ofSEQ ID NO: 10 N199V, K201R, Y213W and S215D or with the following fiveamino acid substitutions: Q193S, N199V, K201R, Y213W and S215D, or maybe found in Table 2 of Kelley et al. (cf. above) and may comprise ahuman TRAIL mutant with the following six amino acid substitutions:Y213W, S215D, Y189A, Q193S, N199V, and K201R, or with Y213W, S215D,Y189A, Q193S, N199R, and K201R.

Examples of mutants of TRAIL with amino acid substitution(s) of theinvention that affect binding and/or activation of TRAILR2 may be found,e.g., in Table 1 of MacFarlane et al. (cf. above) or in Table 2 ofKelley et al. (cf. above) and may comprise a human TRAIL mutant with thefollowing six amino acid substitutions of SEQ ID NO: 10: Y189Q, R191K,Q193R, H264R, I266L, and D267Q, or may be found in Table 2 of van derSloot et al. (cf. above) and may comprise a human TRAIL mutant with thefollowing single amino acid substitution: D269H, or with the followingtwo amino acid substitutions: D269H and E195R or D269H and T214R.

Thus one preferred embodiment is a fusion protein as described hereinwherein at least one of the soluble domains comprises a mutant of TRAILor of a receptor binding domain thereof which binds and/or activatesTRAILR1 and/or TRAILR2.

Further examples of mutants of TRAIL, which show reduced TRAIL inducedreceptor aggregation are H168 (S, T, Q), R170 (E, S, T, Q) and H177 (S,T).

One preferred embodiment of a fusion protein comprising a mutant ofTRAIL or of a receptor binding domain as described herein is a fusionprotein wherein component (i) comprises at least one amino acidsubstitution, particularly as indicated below.

Such an amino acid substitution affects at least one of the followingamino acid positions of human TRAIL (SEQ ID NO: 10): R130, G160, H168,R170, H177, Y189, R191, Q193, E195, N199, K201, Y213, T214, S215, H264,I266, D267, D269.

Such an amino acid substitution is at least one of the following: R130E,G160M, H168 (S, T, Q), R170 (E, S, T, Q), H177 (S,T), Y189A, Y189Q,R191K, Q193S, Q193R, E195R, N199V, N199R, K201R, Y213W, T214R, S215D,H264R, I266L, D267Q, D269H, D269R, or D269K.

A preferred TRAIL-R2 selective domain comprises amino acid substitutionsY189Q, R191K, Q193R, H264R, I266L and D267Q.

A preferred TRAIL-R1 selective domain comprises amino acid substitutionsY189A, Q193S, N199V, K201R, Y213W and S215D.

The single-chain fusion molecule of the present invention comprisesadditionally three soluble cytokine domains, namely components (i),(iii) and (v). According to the present invention, it was surprisinglyfound that the stability of a single-chain TNF family cytokine fusionpolypeptide against aggregation is enhanced, if the second and/or thirdsoluble TNF family cytokine domain is an N-terminally shortened domainwhich optionally comprises amino acid sequence mutations. Thus,preferably, both the second and the third soluble TNF family cytokinedomain are N-terminally shortened domains which optionally compriseamino acid sequence mutations in the N-terminal regions, preferablywithin the first five amino acids of the N-terminus of the solublecytokine domain. These mutations may comprise replacement of charged,e.g. acidic or basic amino acids, by neutral amino acids, particularlyserine or glycine.

In contrast thereto, the selection of the first soluble TNF familycytokine domain is not as critical. Here, a soluble domain having afull-length N-terminal sequence may be used. It should be noted,however, that also the first soluble cytokine domain may have anN-terminally shortened and optionally mutated sequence.

In a preferred embodiment of the present invention, the soluble TNFfamily cytokine domains (i), (iii) and (v) are soluble CD95L domains,particularly soluble human CD95L domains. The first soluble CD95L domain(i) may be selected from native, shortened and/or mutated sequences. TheN-terminal sequence of the first domain (i) may e.g. start between aminoacid Glu142 and Val146 of human CD95L, wherein Arg144 and/or Lys145 maybe replaced by a neutral amino acid, e.g. by Ser or Gly. The second andthird soluble CD95L domains (iii) and (v), however, are selected fromshortened and/or mutated sequences. Preferably, at least one of thesoluble CD95L domains, (iii) and (v), has an N-terminal sequence whichstarts between amino acid Arg144 and Val146 of human CD95L, and whereinArg144 and/or Lys145 may be replaced by a neutral amino acid, e.g. bySer and/or Gly. In an especially preferred embodiment, the second andthird soluble CD95L domain start with an N-terminal sequence selectedfrom:

(a) Arg144-(Gly/Ser) 145-Val (146) (b) (Gly/Ser) 144-Lys145-Val (146)and (c) (Gly/Ser) 144-(Gly/Ser) 145-Val (146).

Further, it is preferred that the CD95L domain ends with amino acid Leu281 of human CD95L.

The soluble CD95L domain may comprise a mammalian, e.g. a humanwild-type sequence. In certain embodiments, however, the CD95L sequencemay comprise a mutation which results in a reduction or completeinhibition of the binding to the extracellular matrix, e.g. a mutationat position Lys177, e.g. Lys177→Glu, Asp or Ser and/or a mutation whichreduces and/or inhibits binding to the CD95L receptor, e.g. a mutationat position Tyr218, e.g. Tyr218→Arg, Lys, Ser, Asp. In certainembodiments of the present invention, one of the three soluble CD95Lmodules is a sequence variant with a reduced receptor binding. In otherembodiments, two of the modules contain mutations resulting in reducedreceptor binding.

In a further preferred embodiment of the present invention, the solubleTNF family cytokine domains (i), (iii) and (v) are soluble TRAILdomains, particularly soluble human TRAIL domains. The first solubleTRAIL domain (i) may be selected from native, shortened and/or mutatedsequences. Thus, the first soluble TRAIL domain (i) has an N-terminalsequence which may start between amino acid Glu116 and Val122 of humanTRAIL, and wherein Arg121 may be replaced by a neutral amino acid, e.g.by Ser or Gly. The second and third soluble TRAIL domains (iii) and (v)have a shortened N-terminal sequence which preferably starts betweenamino acid Gly120 and Val122 of human TRAIL and wherein Arg121 may bereplaced by another amino acid, e.g. Ser or Gly.

Preferably, the N-terminal sequence of the soluble TRAIL domains (iii)and (v) is selected from:

(a) Arg121-Val122-Ala123 and (b) (Gly/Ser)121.

The soluble TRAIL domain preferably ends with amino acid Gly281 of humanTRAIL. In certain embodiments, the TRAIL domain may comprise internalmutations as described above.

In a further preferred embodiment of the present invention, the solubleTNF family cytokine domains (i), (iii) and (v) are soluble LIGHTdomains, particularly soluble human LIGHT domains. The first solubleLIGHT domain (i) may be selected from native, shortened and/or mutatedsequences. Thus, the first soluble LIGHT domain (i) has an N-terminalsequence which may start between amino acid Glu91 and Ala95 of humanLIGHT. The second and third soluble LIGHT domains (iii) and (v) have ashortened N-terminal sequence which preferably starts between amino acidPro94 and Ala95 of human LIGHT. The soluble LIGHT domain preferably endswith amino acid Val240.

Components (ii) and (iv) of the single-chain fusion polypeptide arepeptide linker elements located between components (i) and (iii) or(iii) and (v), respectively. The flexible linker elements have a lengthof 3-8 amino acids, particularly a length of 3, 4, 5, 6, 7, or 8 aminoacids. The linker elements are preferably glycine/serine linkers, i.e.peptide linkers substantially consisting of the amino acids glycine andserine. In cases in which the soluble cytokine domain terminates with Sor G (C-terminus), e.g. human TRAIL, the linker starts after S or G. Incases in which the soluble cytokine domain starts with S or G(N-terminus), the linker ends before this S or G.

It should be noted that linker (ii) and linker (iv) do not need to be ofthe same length. In order to decrease potential immunogenicity, it maybe preferred to use shorter linkers. In addition it turned out thatshorter linkers lead to single chain molecules with reduced tendency toform aggregates. Whereas linkers that are substantially longer than theones disclosed here may exhibit unfavourable aggregations properties.

If desired, the linker may comprise an asparagine residue which may forma glycosylation site Asn-Xaa-Ser. In certain embodiments, one of thelinkers, e.g. linker (ii) or linker (iv) comprises a glycosylation site.In other embodiments, both linkers (iv) comprise glycosylation sites. Inorder to increase the solubility of the scTNF-SF proteins and/or inorder to reduce the potential immunogenicity, it may be preferred thatlinker (ii) or linker (iv) or both comprise a glycosylation site.

Preferred linker sequences are selected from GSGSGSGS (SEQ ID NO:52),GSGSGNGS (SEQ ID NO:53), GGSGSGSG (SEQ ID NO:21), GGSGSG (SEQ ID NO:22),GGSG (SEQ ID NO:23), GGSGNGSG (SEQ ID NO:24), GGNGSGSG (SEQ ID NO:25)and GGNGSG (SEQ ID NO:26)

The fusion protein may additionally comprise an N-terminal signalpeptide domain, which allows processing, e.g. extracellular secretion,in a suitable host cell. Preferably, the N-terminal signal peptidedomain comprises a protease cleavage site, e.g. a signal peptidasecleavage site and thus may be removed after or during expression toobtain the mature protein. Further, the fusion protein may additionallycomprise a C-terminal element, having a length of e.g. 1-50, preferably10-30 amino acids which may include or connect to arecognition/purification domain, e.g. a FLAG domain, a Strep-tag orStrep-tag II domain and/or a poly-His domain.

Further, the fusion polypeptide may additionally comprise N-terminallyand/or C-terminally a further domain, e.g. a targeting domain such as asingle-chain antibody or an antibody fragment domain. Specific examplesof suitable antibodies are anti-tumour antibodies, such as antibodiesagainst EGFR-familiy members. Suitable examples of other targetingmolecules are cytokines, such as interleukins. Examples of specificfusion proteins of the invention are SEQ ID NOs: 27, 28, 29, 43, 45, 47,49 and 51. A further aspect of the present invention relates to anucleic acid molecule encoding a fusion protein as described herein. Thenucleic acid molecule may be a DNA molecule, e.g. a double-stranded orsingle-stranded DNA molecule, or an RNA molecule. The nucleic acidmolecule may encode the fusion protein or a precursor thereof, e.g. apro- or pre-proform of the fusion protein which may comprise a signalsequence or other heterologous amino acid portions for secretion orpurification which are preferably located at the N- and/or C-terminus ofthe fusion protein. The heterologous amino acid portions may be linkedto the first and/or second domain via a protease cleavage site, e.g. aFactor Xa, thrombin or IgA protease cleavage site.

Examples of specific nucleic acid sequences of the invention are SEQ IDNOs: 30, 31 32, 44, 46, 48 and 50.

The nucleic acid molecule may be operatively linked to an expressioncontrol sequence, e.g. an expression control sequence which allowsexpression of the nucleic acid molecule in a desired host cell. Thenucleic acid molecule may be located on a vector, e.g. a plasmid, abacteriophage, a viral vector, a chromosal integration vector, etc.Examples of suitable expression control sequences and vectors aredescribed for example by Sambrook et al. (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, and Ausubel et al. (1989),Current Protocols in Molecular Biology, John Wiley & Sons or more recenteditions thereof.

Various expression vector/host cell systems may be used to express thenucleic acid sequences encoding the fusion proteins of the presentinvention. Suitable host cells include, but are not limited to,prokaryotic cells such as bacteria, e.g. E. coli, eukaryotic host cellssuch as yeast cells, insect cells, plant cells or animal cells,preferably mammalian cells and, more preferably, human cells.

Further, the invention relates to a non-human organism transformed ortransfected with a nucleic acid molecule as described above. Suchtransgenic organisms may be generated by known methods of genetictransfer including homologous recombination.

A further aspect of the present invention relates to a pharmaceutical ordiagnostic composition comprising as the active agent at least onefusion protein, a respective nucleic acid encoding therefore, or atransformed or transfected cell, all as described herein.

At least one fusion protein, respective nucleic acid encoding therefore,or transformed or transfected cell, all as described herein may be usedin therapy, e.g., in the prophylaxis and/or treatment of disorderscaused by, associated with and/or accompanied by dysfunction of TNF-SFcytokines, particularly proliferative disorders, such as tumours, e.g.solid or lymphatic tumours; infectious diseases; inflammatory diseases;metabolic diseases; autoimmune disorders, e.g. rheumatoid and/orarthritic diseases; degenerative diseases, e.g. neurodegenerativediseases such as multiple sclerosis; apoptosis-associated diseases ortransplant rejections.

The term “dysfunction of TNF-SF cytokines” as used herein is to beunderstood as any function or expression of a TNF-SF cytokine thatdeviates from the normal function or expression of a TNF-SF cytokine,e.g., overexpression of the TNF-SF gene or protein, reduced or abolishedexpression of the TNF-SF cytokine gene or protein compared to the normalphysiological expression level of said TNF-SF cytokine, increasedactivity of the TNF-SF cytokine, reduced or abolished activity of theTNF-SF cytokine, increased binding of the TNF-SF cytokine to any bindingpartners, e.g., to a receptor, particularly a CD95 or TRAIL receptor oranother cytokine molecule, reduced or abolished binding to any bindingpartner, e.g. to a receptor, particularly a CD95 or TRAIL receptor oranother cytokine molecule, compared to the normal physiological activityor binding of said TNF-SF cytokine.

The composition may be administered as monotherapy or as combinationtherapy with further medications, e.g. cytostatic or chemotherapeuticagents, corticosteroids and/or antibiotics.

The fusion protein is administered to a subject in need thereof,particularly a human patient, in a sufficient dose for the treatment ofthe specific conditions by suitable means. For example, the fusionprotein may be formulated as a pharmaceutical composition together withpharmaceutically acceptable carriers, diluents and/or adjuvants.Therapeutic efficacy and toxicity may be determined according tostandard protocols. The pharmaceutical composition may be administeredsystemically, e.g. intraperitoneally, intramuscularly or intravenouslyor locally, e.g. intranasally, subcutaneously or intrathecally.Preferred is intravenous administration.

The dose of the fusion protein administered will of course be dependenton the subject to be treated, on the subject's weight, the type andseverity of the disease, the manner of administration and the judgementof the prescribing physician. For the administration of fusion proteins,a daily dose of 0.001 to 100 mg/kg is suitable.

EXAMPLES 1. Manufacture of a Single-Chain CD95L Fusion Protein (scCD95L)

In the following, the general structure of the recombinant proteins ofthe invention (FIG. 1) is shown exemplified for the receptor bindingdomain of the human CD95 ligand.

1.1 Polypeptide Structure

-   -   A) Amino acids Met1-Ser21        -   IgKappa-signal peptide, assumed signal peptidase cleavage            site after amino acid Gly20    -   B) Amino acids Glu22-Leu161        -   First soluble cytokine domain of the human CD95 ligand            (CD95L; amino acids 142-281 of SEQ ID NO: 6 including a            K145S mutation).    -   C) Amino acids Gly162-G1y169        -   First peptide linker element.    -   D) Amino acids Arg170-Leu307        -   Second soluble cytokine domain of the human CD95 ligand            (CD95L; amino acids 144-182 of SEQ ID NO: 6 including a            K145S mutation).    -   E) Amino acids Gly308-315        -   Second peptide linker element.    -   F) Amino acids Arg316-Leu453        -   Third soluble cytokine domain of the human CD95 ligand            (CD95L; amino acids 144-281 of SEQ ID NO: 6 including a            K145S mutation).    -   G) Amino acid Gly457-Lys472        -   Peptide linker with a Strep-tag II motif.

The amino acid sequence of sc CD95L is shown in SEQ ID NO. 27. Thefusion polypeptide comprises first and second peptide linkers having thesequence GGSGSGSG (SEQ ID NO: 21). Further preferred linker sequencesare SEQ ID NOs: 22-26 as described above. It should be noted that thefirst and second peptide linker sequences need not to be identical.

The signal peptide sequence (A) may be replaced by any other suitable,e.g. mammalian signal peptide sequence. The Strep-tag II motif (G) maybe replaced by other motifs, if desired, or deleted.

As shown in FIG. 23, cell culture supernatant of HEK293 cells,transiently expressing scCD95L (SEQ ID NO:27) was collected and used tostimulate Jurkat cells at varying concentrations. The supernatant wasused either directly without further modifications or an anti-Streptagantibody (2 microgram/ml) was added to cross-link the scCD95L protein.Only cell supernatant that contained cross-linked scCD95L-St increasedcaspase activity in Jurkat cells, indicating that scCD95L alone does notform higher order aggregates able to be pro-apoptotic.

1.2 Gene Cassette Encoding the Polypeptide

The synthetic gene may be optimised in view of its codon-usage for theexpression in suitable host cells, e.g. insect cells or mammalian cells.A preferred nucleic acid sequence is shown in SEQ ID NO: 30.

1.3 Cloning Strategy

The synthetic gene may be cloned, e.g. by means of a restriction enzymehydrolysis into a suitable expression vector.

2. Manufacture of a Single-Chain TRAIL Fusion Protein (sc TRAIL wt) 2.1Polypeptide Structure A) Amino Acids Met1-Gly20

-   -   Ig-Kappa-signal peptide, assumed signal peptidase cleavage site        after amino acid Gly 20.

B) Amino Acids GIn21-Gly182

-   -   First soluble cytokine domain of the human TRAIL ligand (TRAIL,        amino acid 120-281 of SEQ ID NO:10)

C) Amino Acids Gly183-Ser 190

-   -   First peptide linker element, wherein the two amino acids        designated X are both S or one is S and the other one is N.

D) Amino Acids Arg191-Gly351

-   -   Second soluble cytokine domain of the human TRAIL ligand (TRAIL,        amino acids 121-281 of SEQ ID NO:10)

E) Amino Acids Gly 352-Ser359

-   -   Second peptide linker element wherein the two amino acids        designated X are both S or one is S and the other one is N.

F) Amino Acids Arg360-Gly520

-   -   Third soluble cytokine domain of the human TRAIL ligand (TRAIL,        amino acids 121-Gly281 of SEQ ID NO:10).

G) Amino Acids Gly521-Lys538

-   -   Peptide linker element with a Streptag II motif.

The amino acid sequence of sc TRAIL wt is shown in SEQ ID NO: 28.

The indicated linkers may be replaced by other preferred linkers, e.g.as shown in SEQ ID NOs: 21.26. It should be noted that the first andsecond peptide linkers do not need to be identical.

The signal peptide sequence (A) may be replaced by any other suitable,e.g. mammalian signal peptide sequence. The Strep-tag II motif (G) maybe replaced by other motifs, if desired, or deleted.

Cell culture supernatants of HEK293 cells, transiently expressing singlechain TRAIL proteins with different linkers (derived from SEQ ID 28, intotal nine different linker combinations) were collected and used tostimulate Jurkat cells at varying dilutions (exemplarily, a dilution of1:8 is shown in FIG. 25). The supernatants were used either directlywithout further modifications or an anti-Streptag antibody (2microgram/ml Strep MAB Immo) was added to cross-link the scTRAILwtproteins. Jurkat cells were incubated with HEK293 cell culturesupernatant for three hours at 37°, lysed and analysed for caspaseactivity. Cell culture supernatant that contained cross-linked scTRAILwtproteins induced an increased caspase activity in Jurkat cells (resultsshown on the right hand side of the graph), indicating that scTRAILwtproteins alone do form only a low amount of higher order aggregates ableto be pro-apoptotic.

2.2 Gene Cassette Encoding the Polypeptide

The synthetic gene may be optimised in view of its codon usage for theexpression in suitable host cells, e.g. insect cells or mammalian cells.A preferred nucleic acid sequence is shown in SEQ ID NO: 31.

3. Manufacture of a Single-Chain Mutated TRAIL Fusion Protein (scTRAIL(R2-Specific))

In the following, the structure of a single-chain TRAIL polypeptidecomprising a mutation for selective binding to TRAIL receptor R2 isshown.

3.1 Polypeptide Structure A) Amino Acids Met1-Ser29

-   -   Ig-Kappa signal peptide, assumed signal peptidase cleavage site        after amino acid Gly20 and peptide linker

B) Amino Acids Arg29-Gly190

-   -   First soluble cytokine domain of the human TRAIL ligand (TRAIL,        amino acids 121-281 of SEQ ID NO: 10 including the mutations        Y189Q, R191K, Q193R, H264R, I266L and D267Q)

C) Amino Acid Gly191-Ser198

-   -   First peptide linker element, wherein the amino acids designated        X are as indicated in Example 2

D) Amino Acids Arg199-Gly359

-   -   Second soluble cytokine domain of the human TRAIL ligand (TRAIL        amino acids 121-281 of SEQ ID NO: 10 including the mutations as        indicated in B)

E) Amino Acids Gly360-Ser367

-   -   Second peptide linker element, wherein the amino acids X are as        indicated in Example 2

F) Amino Acids Arg368-Gly528

-   -   Third soluble cytokine domain of the human TRAIL ligand (TRAIL,        amino acids 121-281 of SEQ ID NO: 10 including the mutations as        indicated in B)

G) Amino Acids Gly529-Lys546

-   -   Peptide linker with a Strep-tag II motif

The amino acid sequence of scTRAIL(R2-specific) is shown in SEQ ID NO:29.

The indicated linkers may be replaced by other preferred linkers, e.g.as shown in SEQ ID NOs: 21-26. It should be noted that the first andsecond peptide linkers do not need to be identical.

The signal peptide sequence (A) may be replaced by any other suitable,e.g. mammalian signal peptide sequence. The Streptag II motif (G) may bereplaced by other motifs, if desired, or deleted.

3.2 Gene Cassette Encoding the Polypeptide

The synthetic gene may be optimised in view of its codon usage for theexpression in suitable host cells, e.g. insect cells or mammalian cells.A preferred nucleic acid sequence is shown in SEQ ID NO: 32.

4. Expression and Purification a) Cloning, Expression and Purificationof Fusion Polypeptides

Hek293T cells grown in DMEM+GlutaMAX (GibCo) supplemented with 10% FBS,100 units/ml Penicillin and 100 μg/ml Streptomycin were transientlytransfected with a plasmid containing an expression cassette for afusion polypeptide. In those cases, where a plurality of polypeptidechains is necessary to achieve the final product, e.g. for theFab-scTNF-SF fusion proteins (FIG. 9A), the expression cassettes wereeither combined on one plasmid or positioned on different plasmidsduring the transfection. Cell culture supernatant containing recombinantfusion polypeptide was harvested three days post transfection andclarified by centrifugation at 300×g followed by filtration through a0.22 μm sterile filter. For affinity purification Streptactin Sepharosewas packed to a column (gel bed 1 ml), equilibrated with 15 ml buffer W(100 mM Tris-HCl, 150 mM NaCl, pH 8.0) or PBS pH 7.4 and the cellculture supernatant was applied to the column with a flow rate of 4ml/min. Subsequently, the column was washed with 15 ml buffer W andbound polypeptide was eluted stepwise by addition of 7×1 ml buffer E(100 mM Tris HCl, 150 mM NaCl, 2.5 mM Desthiobiotin, pH 8.0).Alternately, PBS pH 7.4 containing 2.5 mM Desthiobiotin can be used forthis step. The protein amount of the eluate fractions was quantitatedand peak fractions were concentrated by ultrafiltration and furtherpurified by size exclusion chromatography (SEC).

SEC was performed on a Superdex 200 column using an Äkta chromatographysystem (GE-Healthcare). The column was equilibrated with phosphatebuffered saline and the concentrated, Streptactin-purified polypeptidewas loaded onto the SEC column at a flow rate of 0.5 ml/min. The elutionprofile of the polypeptide was monitored by absorbance at 280 nm.

For determination of the apparent molecular weight of purified fusionpolypeptide under native conditions a Superdex 200 column was loadedwith standard proteins of known molecular weight. Based on the elutionvolume of the standard proteins a calibration curve was plotted and theapparent molecular weight of purified fusion polypeptide was determined.

5, Apoptosis Assay

A cellular assay with a Jurkat A3 permanent T-cell line was used todetermine the apoptosis inducing activity of different CD95-ligand(CD95L) and TRAIL fusion polypeptide constructs. Jurkat cells were grownin flasks with RPMI 1640-medium+GlutaMAX (GibCo) supplemented with 10%FBS, 100 units/ml Penicillin and 100 μg/ml Streptomycin. Prior to theassay, 100,000 cells were seeded per well into a 96-wellmicrotiterplate. The addition of different concentrations of fusionpeptides to the wells was followed by a 3 hour incubation at 37° C.Cells were lysed by adding lysis buffer (250 mM HEPES, 50 mM MgCl₂, 10mM EGTA, 5% Triton-X-100, 100 mM DTT, 10 mM AEBSF, pH 7.5) and plateswere put on ice for 30 minutes to 2 hours. Apoptosis is paralleled by anincreased activity of caspases, e.g. Caspase-3. Hence, cleavage of thespecific caspase substrate Ac-DEVD-AFC (Biomol) was used to determinethe extent of apoptosis. In fact, Caspase activity correlates with thepercentage of apoptotic cells determined morphologically after stainingthe cells with propidium iodide and Hoechst-33342. For the caspaseactivity assay, 20 μl cell lysate was transferred to a black 96-wellmicrotiterplate. After the addition of 80 μl buffer containing 50 mMHEPES, 1% Sucrose, 0.1% CHAPS, 50 μM Ac-DEVD-AFC, and 25 mM DTT, pH 7.5,the plate was transferred to a Tecan Infinite 500 microtiterplate readerand the increase in fluorescence intensity was monitored (excitationwavelength 400 nm, emission wavelength 505 nm).

5.1 Cell Death Assay

For the determination of cell death in HT1080 fibrosarcoma cells 15,000cells were plated in 96-well plates over night in RPMI1640-medium+GlutaMAX (GibCo) supplemented with 10% FBS (Biochrom). Cellswere coincubated with cycloheximide (Sigma) at a final concentration of2.5 g/ml. Cell death was quantified by staining with buffer KV (0.5%crystal violet, 20% methanol). After staining, the wells were washedwith water and air-dried. The dye was eluted with methanol and opticaldensity at 595 nm was measured with an ELISA reader.

6. Stability/Aggregation Test 6.1. Principle of the Aggregation Analysis(Definition for Soluble Protein)

The content of monomers (defined trimeric assembly of TNF-SF receptorbinding modules) and aggregates is determined by analytical SEC asdescribed in Example 4. For this particular purpose the analysis isperformed in buffers containing physiological salt concentrations atphysiological pH (e.g. 0.9% NaCl, pH 7.4; PBS pH 7.4). A typicalaggregation analysis is done on a Superdex200 column (GE Healthcare).This column separates proteins in the range between 10 to 800 kDa.

For determination of the apparent molecular weight of purified fusionpolypeptide under native conditions a Superdex 200 column is loaded withstandard proteins of known molecular weight. Based on the elution volumeof the standard proteins a calibration curve is plotted and the apparentmolecular weight of purified fusion polypeptide is calculated based onthe elution volume.

SEC analysis of soluble, non aggregated protein s, —e.g. trimericTNF-SF, typically shows a distinct single protein peak at a definedelution volume. This elution volume corresponds to the apparent nativemolecular weight of the particular protein and approximately complies tothe theoretical molecular weight calculated on the basis of the primaryamino acid sequence.

If protein aggregation occurs the SEC analysis shows additional proteinpeaks with lower retention volumes. For TNF-SF family members theaggregation of soluble proteins occurs in a characteristic manner. Theproteins tend to form oligomers of the “trimers”, forming nonamers (3×3)and 27mers (3×9). These oligomers serve as aggregation seeds and a highcontent of oligomers potentially leads to aggregation of the protein.Oligomers of large molecular weight and aggregates elute in the voidvolume of the Superdex200 column and cannot be analysed by SEC withrespect to their native molecular weight. Examples for SEC analysis of adefined soluble trimeric and a oligomerised/aggregated preparation ofTNF-SF proteins are shown in FIG. 17.

Due to the induction of (complete) aggregation, purified preparations ofTNF-SF fusion proteins should preferably contain only defined trimericproteins and only a very low amount of oligomerised protein.

The degree of aggregation/oligomerisation of a particular TNF-SF proteinpreparation is determined on basis of the SEC analysis by calculatingthe peak areas of the OD280 diagram for the defined trimeric and theoligomer/aggregate fraction, respectively. Based on the total peak areathe percentage of defined trimeric protein is calculated as follows:

(% Trimer content=[Peak area trimer]/[Total peak area]×100)

The definition for soluble protein as used in this text, describes aprotein preparation of purified TNF-SF protein in a buffer ofphysiological salt concentrations at physiological pH that contains adefined soluble protein (trimeric assembly of TNF-SF domains) contentof >90% within a typical protein concentration range from 0.2 to 10.0mg/ml.

6.2 SEC Aggregation Analysis for Purified sc-TRAIL Variants

Three different sc-TRAIL variants were transfected and affinity purifiedas described. The purified proteins were subsequently analysed for theircontent of defined soluble protein using SEC analysis as described in6.1. In the particular case of single chain fusion proteins a trimerdescribes a trimeric assembly of three encoded TNF-SF domains encoded bya single polypeptide chain. (Formally single chain TNF-SF proteins aremonomers, since single chain assemblies do only form intramolecularinteractions [all protein domains are encoded by a single polypeptidechain] and do not form intermolecular interactions between distinctindividual polypeptide chains.)

The proteins analysed by SEC were:

-   -   1.) Fab-sc-TRAIL(R2-specific)-SNSN (FIG. 19):    -   Fusion protein comprising an Fab domain fused N-terminal to a        single chain fusion protein of TRAIL specific for TRAIL-receptor        2 interaction, glycosylated    -   2.) Fab-sc-TRAIL(R2-specific)-SSSS (FIG. 18)    -   Fusion protein comprising an Fab domain fused N-terminal to a        single chain fusion protein of TRAIL specific for TRAIL-receptor        2 interaction, non glycosylated    -   3.) Fab-sc-TRAIL-wt-SNSN (FIG. 20):    -   Fusion protein comprising an Fab domain fused N-terminal to a        single chain TRAIL, glycosylated

The SEC analysis for the three purified Fab-sc-constructs of TRAILrevealed a single protein peak for all proteins indicating definedsoluble protein fractions (>95% trimer). The calculated apparent MW forthe proteins (based on calibration of the column) strongly indicate atrimeric association of the TNF-SF-domains for the purified proteins.None of the analysed proteins showed indications for aggregation (FIGS.18, 19, 20).

Comparing the potentially glycosylated “Fab-sc-TRAIL-R2-SNSN” with thenon glycolsylated “Fab-sc-TRAIL-R2-SSSS” indicates a significantdifference of the apparent native MW that is due to glycosylation ofFab-sc-TRAIL(R2-specific)-SNSN.

Expression of sc-TNF-SF members as fusion protein with an antibodyfv-fragment is known to facilitate aggregation of the protein. Theconstruction principle of the Fab-sc-TRAIL variants revealed noaggregation of the expressed TRAIL variants and is therefore beneficialwith respect to solubility of the protein.

6.3 Differential Glycolsylation of sc-TRAIL-Linker Variants

Glycosylation of proteins can be beneficial for recombinant sc-TNF-SFconstructs with regard to potential immunogenicity and stability. Inorder to get glycosylation of the sc-TRAIL construct, specific linkersequences were designed that contained putative N-linked glycosylationsites at defined positions (see FIG. 21-A). Recombinant expression andsubsequent Western-Blot analysis revealed that the respective positionof the Asparagine (N) within the linker sequence is important for thesubsequent glycosylation of the protein. Surprisingly, the preferentiallinker position of the glycosylated asparagine was identified to be atposition “2” as described in FIG. 21-A, (G S G S G N G S). If theasparagine is localised at other positions (e.g. position “1” [G S G N GS G S] see FIG. 21-A), glycosylation of the respective asparagines(s) isabolished. This aspect could be confirmed by Western-Blot analysis ofdifferent sc-TRAIL variants. If both asparagines of linker 1 and linker2 were localised at position“2” a significant glycosylation dependantMW-shift could be observed for the respective sc-TRAIL variant (FIG.22). A MW-shift of the glycosylated sc-TRAIL linker variant could alsobe confirmed by calculating the apparent MW after SEC analysis (FIG. 18,19). The non glycosylated Fab-sc-TRAIL(R2-specific)SSSS has a clearlylower MW (68 kDa) compared to glycosylated Fab-sc-TRAIL(R2-specific)SNSN(87 kDa).

Based on this analysis we claim differential glycosylation of thesc-TRAIL constructs by modifying the position of the asparagines withinthe linker sequence(s). Glycosylation protects the linker sequencetowards proteolytic degradation and might stabilise the protein. Inaddition glycosylation of the linker sequence potentially preventsrecognition of the linker sequence by the immune system and potentiallyreduces the immunogenicity of the protein. Therefore glycosylation ofthe linker sequence is beneficial with regard to immunogenicity andproteolytic stability of the sc-TRAIL constructs and has potentialinfluence on the half life of the protein. The linker specificdifferential glycosylation can be used to modify the immunogenicity andstability of recombinant TNF-SF members.

6.3. Expression and Analysis of a sc-TRAIL with Prolonged LinkerSequence and N-Terminal Stalk Residues (sc-TRAIL-(95.281)-long)

In WO/2005/103077 a single chain TRAIL-fusion polypeptide, herein namedsc-TRAIL-(95-281)-long, is described, wherein each TRAIL module compriseresidues 95 to 281 of SEQ ID NO:10. The TRAIL modules are linked byGlycin Serin linker comprising of at least 12 amino acids(GGGSGGGSGGGS). Compared to the TRAIL modules of the present invention(comprising residues 121-281 of SEQ ID NO:10), additional 25 amino acidsincluding the stalk region are present in each of the adjacant TRAILmodules.

In order to analyse the influence of the linker sequence on sc-TRIALconstructs, sc-TRAIL-(95-281)-long is analysed. Expression, purificationand subsequent SEC analysis reveals that sc-TRAIL-(95-281)-long with the12 aa linker and the additional stalk sequence is expressed and secretedto the cell culture supernatant of HEK293T cells. However, SEC analysisof the purified protein indicates that sc-TRAIL-(95-281)-long showsmultiple peaks comprising a large amount of protein in an oligomerisedor aggregated from. Aggregation of sc-TRAIL-(95-281)-long is a directeffect of the prolonged linker sequences in combination with theadditional residues of the N-terminal stalk. The results indicate thatthe longer linker used in this construct leads to increased aggregationproperties of the construct.

7. Construction of Single-Chain Fusion Polypeptides Comprising One orMore Additional Domains

7.1. Assembly of Soluble TNF-SF and Antibody Fragments Known from theArt

It is known from the art that soluble TNF-SF cytokine domains may befused to antibody fragments in order to obtain trimerisation and/ordimerisation of trimers. Single-chain scFv-TNF-SF fusion proteins havebeen constructed consisting of a single-chain antibody and a solubledomain comprising a TNF-RBD and the stalk-region. The correspondingtrimers consist of three single-chain antibodies and three solubledomains (FIG. 7).

In addition, Fc-TNF-SF fusion proteins, wherein each fusion proteincomprises an N-terminal intramolecular Fc-domain and a C-terminalsoluble domain have been constructed (FIG. 8). The dimerisation ofsoluble domains is accomplished by assembly of two Fc-domains viadisulfide bridges. Trimers are subsequently obtained by a combination oftwo soluble domains from one Fc-TNF-SF fusion protein and one solubledomain from another Fc-TNF-SF fusion protein. As can be deduced fromFIG. 4, dimerisation of trimers is also mediated by the N-terminalFc-TNF-SF fusion. In conclusion, three Fc-antibody fragments are presentper dimer of the trimer. However, such fusion proteins are likely toform higher molecular weight aggregates, which represents a majordisadvantage.

7.2 Fusion Proteins of the Invention Comprising One or More AdditionalDomains

The inventive fusion proteins comprising one or more additional domainscan be constructed in several ways. In the following, the constructionof fusion proteins with additional domains is exemplified with theantibody pertuzumab directed against the cell surface antigen ErbB2.

The amino acid sequence of the heavy chain is shown in SEQ ID NO: 33:

  1 EVQLVESGGG LVQPGGSLRL SCAASGFTFT DYTMDWVRQA PGKGLEWVAD VNPNSGGSIY 61 NQRFKGRFTL SVDRSKNTLY LQMNSLRAED TAVYYCARNL GPSFYFDYWG QGTLVTVSSA121 STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG181 LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK  SC

The amino acid sequence of the light chain is shown in SEQ ID NO: 34

  1 DIQMTQSPSS LSASVGDRVT ITCKASQDVS IGVAWYQQKP GKAPKLLIYS ASYRYTGVPS 61 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YYIYPYTFGQ GTKVEIKRTV AAPSVFIFPP121 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT181 LSKADYEKHK vyACEVTHQG LSSPVTKSFN RGEC7.2.1

In one embodiment, the fusion polypeptide of the invention furthercomprises an N- or C-terminal Fab-antibody fragment (FIG. 9A).

The fusion of an antibody Fab-fragment to the N-terminus of scTNF-SFfusion polypeptide may be accomplished by the following two strategies:

(i) The heavy chain sequence is extended by further amino acids from theIgG1 hinge region and fused to the single-chain TNF-SF fusion protein.

The IgG1 hinge region comprises the amino acid sequence SEQ ID NO: 35:

. . . KS

₁DKTHT

PP

PAPE . . .

In a preferred embodiment, the Fab-domain is chosen such that theC-terminal cysteine of the heavy chain (C1 of the hinge region)terminates the CH1 domain. This cysteine is required for forming adisulfide linkage to the light chain.

The subsequent linker comprises portions of the IgG hinge region (e.g.DKTHT or DKT), however without further cysteines of the hinge region.Alternatively, a glycine/serine linker is used. Due to the absence offurther cysteines, a monomeric fusion protein comprising two polypeptidechains is obtained. The linker preferably has a length of 3-15 aminoacids. More preferably, the linker is selected from the linker 1-7 asshown below.

1. DKTHTG(S)a(G)b; (a = 0-5; b = 0 oder 1) 2.DKTHTGS(S)a(GS)bG(S)c (a, b = 0, 1-6; c = 0 oder 1) 3.DKTG(S)a(G)b; (a=/ 0-5; b = 0 oder 1) 4. DKTG(S)a(GS)bG(S)c (a, b =0, 1-6; c = 0 oder 1) 5. SSG(S)a(GS)bG(S)c (a, b = 0, 1-6; c = 0 oder 1)6. SS(GGGS)aG(S)b (a = 0, 1-4; b = 0  oder 1) 7. GSPGSSSSSS(G)a (a =0 oder

Preferred amino acid sequences with the heavy chain module positionedN-terminal to the scTNF-SF module are shown in SEQ ID NO: 45, SEQ ID NO:47 and SEQ ID NO: 49. For production purposes, these polypeptide chainsare coexpressed with the Fab light chain polypeptide (SEQ ID NO: 40) tofinally achieve the Fab-scTRAIL fusion polypeptides.

(ii) The light-chain sequence is fused to the single chain TNF-SF fusionprotein.

The constant region of the light chain (e.g. SEQ ID NO: 34) ends with aC-terminal cysteine residue. This residue may be covalently bridged withthe C1 hinge cysteine of the heavy chain. Preferably, the linkers 1-7 asshown below are used for the connection between the light chain sequenceand the TNF-SF fusion protein. Linkers 5-7 are preferred (see above).

Preferably, the last amino acid in the linker adjacant to the cytokinemodule is either Gly or Ser. In the following, preferred linkersequences are shown:

Further, the linker may comprise N-glycosylation motifs (NXSIT, whereinX may be any amino acid). One embodiment of the amino acid sequenceswith the light chain module positioned N-terminal to the scTNF-SF moduleis shown in SEQ ID NO: 51.

In the case of the Fab-scTNF-SF fusion proteins, the co-expression oftwo polypeptide chains is necessary to achieve the correct assembly ofthe Fab module in addition to the scTNF-SF module (see FIG. 9A). ThePertuzumab heavy and light chain modules (SEQ ID NO: 33 and SEQ ID NO:34) were equipped with a signal peptide, backtranslated and theresulting synthetic genes (SEQ ID NO: 41 and SEQ ID NO: 42) geneticallyfused upstream of the scTRAILwt- or scTRAILR2-specific gene modules (SEQID NO: 31 and SEQ ID NO: 32). Examples for the resulting gene cassettesare shown in SEQ ID NO: 46, 48 and 50. After subcloning into appropriateexpression vectors, a selection of the resulting plasmids was used fortransient protein expression in HEK293T cells. The heavy chain TRAIL orlight chain TRAIL expression plasmids were transfected either alone orin combination with the necessary light or heavy chain encoding vectorsof the Fab-Fragment (FIG. 26). Surprisingly, the module combinationwithin the fusion proteins influenced the relative stability of thescTRAIL-protein during secretory based expression. If the light-chainmodule of the Fab-domain is fused N-terminal to the scTRAIL-domain(exemplified in SEQ ID NO: 51), the expression product is stable itselfand secreted, when expressed separately (Lanes 1-4, FIG. 26). It can betherefore expected, when such a fusion polypeptide is coexpressed with aheavy-chain module, that two major protein species will be formed duringa potential production process: (1) the Fab-scTRAIL fusion proteinconsisting of two polypeptide chains and (2) as contamination alight-chain-scTRAIL fusion protein without a functional Fab domain.Therefore, fusing the heavy-chain module N-terminal to thescTNF-SF-module for the expression is preferred to avoid this technicaldisadvantage.

A functional analysis of recombinant inventive Fab comprising-scTRAILfusion proteins with the heavy-chain module fused N-terminal to thescTRAIL-module (Fab-scTRAILR2-SNSN or Fab-scTRAILwt-SNSN) is shown inFIG. 28. As final purification step, size exclusion chromatography wasemployed as exemplified in FIGS. 19 and 20.

Superior bioactivity compared to soluble, homotrimeric ligands caneasily be achieved by the use of artificially cross-linked or amembrane-bound ligand of the TNF superfamily. Thus the local enrichmentof single chain TRAIL (scTRAIL) constructs on cells that express theantigen Her2 via the Her2-selective Fab-fragment (“Pertuzumab”) fused tothese scTRAIL proteins should increase their cytotoxic bioactivity.Likewise, the blocking of the Her2 binding sites on cells bypre-incubation with the Her2-specific Fab-fragment (Pertuzumab-Fab) onlyshould decrease the cytotoxic bioactivity of Fab-scTRAIL fusionproteins. As shown in FIG. 28A, scTRAIL constructs induce the death ofHT1080 cells, as the viability decreases with increasing proteinconcentration. In accordance, the pre-incubation of HT1080 cells withthe Fab-fragment (Pertuzumab-Fab), followed by co-incubation with theFab-scTRAIL constructs (Fab-scTRAILR2-SNSN or Fab-scTRAILwt-SNSN) overnight, reduced the cytotoxic activity of the Fab-scTRAIL constructs(FIG. 28B), whereas the Fab only induced no cell death.

An increased technical effect may be achieved by use of artificiallycross-linked or a membrane-bound ligands of the TNF superfamilyresulting especially in superior bioactivity as compared to soluble,homotrimeric ligand. Thus the local enrichment of ligands or singlechain ligands such as exemplified by single chain TRAIL (scTRAIL) oncells or on neighbouring cells should increase the bioactivity of thesefusion proteins. The local enrichment (or targeting) of these singlechain ligands can be specifically induced for instance by fusing thesingle chain ligands with amino acid sequences that bind to any antigenpresent on cells such as for instance tumor cells. Examples for antigenbinding sequences may be derived from antibodies such as scFv or Fabfragments. Examples for antigens expressed on target cells may bereceptors such as from the EGFR family or any other antigen to which abinding antibody can be generated. Of special interest in this contextare cell surface antigens specific for tumor or cancer cells.

7.2.2

In another embodiment, the fusion polypeptide of the invention furthercomprises an additional N- or C-terminal scFv-antibody fragment (FIG.9B).

In this embodiment linkers 5-7 as described above may be used. Further,the linkers may comprise N-glycosylation motifs.

A preferred single chain Fv-pertuzumab fragment for fusing to thesingle-chain cytokine fusion protein may comprise amino acidsGlu1-Ser119 of SEQ ID NO: 33 and Asp-Lys107 or Thr109 of SEQ ID NO: 34.The VH and VL fragments may be connected by a linker.

One embodiment of a scFv-domain of pertuzumab is shown in the followingSEQ ID NO: 36:

  1 METDTLLLWV LLLWVPAGNG EVQLVESGGG LVQPGGSLRL SCAASGFTFT DYTMDWVRQA 61 PGKGLEWVAD VNPNSGGSIY NQRFKGRFTL SVDRSKNTLY LQMNSLRAED TAVYYCARNL121 GPSFYFDYWG QGTLVTVSSG GGGSGGGGSG GGGSDIQMTQ SPSSLSASVG DRVTITCKAS181 QDVSIGVAWY QQKPGKAPKL LIYSASYRYT GVPSRFSGSG SGTDFTLTIS SLQPEDFATY241 YCQQYYIYPY TFGQGTKVEI KRT

Amino acids 1-20 (underlined) constitute an N-terminal secretory signalpeptide.

7.2.3

In a further embodiment, the fusion polypeptide of the inventioncomprises an additional N- or C-terminal Fc-antibody fragment (FIGS. 10and 11).

Preferably, the Fc-antibody fragment domain is derived from a humanimmunoglobulin G heavy chain, particularly from a human immunoglobulinIgG1 heavy chain. In an especially preferred embodiment, the amino acidsequence of the Fc-domain is shown in SEQ ID NO: 37.

  1 KS C DKTHT C P P C PAPELLGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS HEDPEVKFNW  61 YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS 121 KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV 181 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYTQKSLSLSPGK

Amino acids Lys1-Glu16 define the hinge region.

For a C-terminal fusion (FIG. 11) the Fc-domain preferably comprises thecomplete constant domain (amino acids 17-230 of SEQ ID NO: 37) and apart or the complete hinge region, e.g. the complete hinge region or thehinge region starting from amino acid Asp4.

Preferred linkers for connecting a C-terminal Fc-antibody fragment (e.g.FIG. 11) are shown in the following:

Linker 8 scCD95L/scTRAIL . . . GG(P/S)_(a)(GS)_(b)(G/S)_(c)KSCDKTHTCPPCPAPE . . . (a = 0 oder 1; b = 0-8; c = 0-8) Linker 9scCD95L/scTRAIL . . . GG(P/S)_(a)(GSSGS)_(b) GS(G/S)_(c)DKTHTCPPCPAPE . . . (a = 0 oder 1; b = 0-8; c = 0-8) Linker 10scCD95L/scTRAIL . . . GG(P/S)_(a)(S)_(b)(GS)_(c)(G/S)_(d)DKTHTCPPCPAPE . . . (a = 0 oder 1; b = 0-8; c = 0-8; d = 0-8)

All linkers start with GlyGly taking in account, however, that theC-terminal amino acid of TRAIL is a Gly. At position 3 of the linker,alternatively Pro or Ser are present. Linker 8 comprises the Cys1cysteine of the heavy chain.

It should be noted that linkers 8-10 are also suitable for theC-terminal fusion of other polypeptides, e.g. a further scTNF-SF fusionprotein.

In detail, the scTRAILwt module (SEQ ID NO: 28), thescTRAIL(R2-specific)-module (SEQ ID NO: 29) and the scCD95L-module (SEQID NO: 27) were fused N-terminally to the Fc-domain of human IgG1,starting with Asp4 of SEQ ID NO: 37 employing four linker elements asshown in table 2.

Fc- Amino-acid sequence Fusion of the linker element FC01. . . (G)GSPGSSSSSSGSDKTH . . . FC02 . . . (G)GSPGSSSSGSDKTH . . . FC03. . . (G)GSPGSSGSDKTH . . . FC04 . . . (G)GSSDKTH . . .

Table 2: Sequences linking the Fc-domain C-terminally to scTNF-SFmodule. The N-terminal amino-acid of the IgG1 CH2-domain is underlined.The N-terminal Glycine of the linking sequence is shown in brackets. ForTNF-SF proteins with a glycine as the C-terminal amino acid (e.g.TRAIL), the N-terminal glycine of the linking sequence formally belongsto the scTNF-SF module.

For purification and characterisation, a Strep-tag II (amino acidsequence WSHPQFEK) was placed C-terminally to the Fc-domain. Thisaffinity tag was linked to the CH3-domain by a flexible linker element(amino acid sequence SSSSSSA), replacing the C-terminal lysine residueof the CH3-sequence. The amino acid sequences of the scTNF-SF fusionproteins as well as for the described protein modules werebacktranslated and their codon usage was optimised for mammaliancell-based expression, Gene synthesis was done by ENTELECHON GmbH(Regensburg, Germany). The expression cassettes for larger fusionproteins were assembled by common cloning procedures starting withDNA-modules of suitable size and suitable restriction enzyme pattern.Exemplarily, the resulting gene cassette for the single chain TRAILwtFC01 fusion protein (scTRAILwt-FC01) is shown in SEQ ID NO: 44 and theencoded protein sequence is shown in SEQ ID NO: 43. The gene cassettesencoding the shortened linker variants (table 1) were generated by PCRbased subcloning strategies, starting from SEQ ID NO: 44. The finalexpression cassettes were released from intermediate cloning vectors andsubcloned into to pcDNA4-HisMax-backbone, using unique Hind-III-, Not-I-or Xba-I sites of the plasmid. For the assembly of the Fab- andFc-fusions proteins, a unique SgS-I site was introduced into the vectorbackbone, replacing the Not-I-site. All expression cassettes wereroutinely verified by DNA sequencing. The proteins were transientlyexpressed in HEK293T cells and the cell culture supernatants weremonitored regarding their pro-apoptotic activity. As shown in FIG. 27,the scTRAIL-Fc fusion proteins of the invention, were able to induce apronounced increase in caspase activity, confirming the potency of theFc-based dimerisation of two scTRAILwt-modules. Similar results wereobtained for scTRAIL(R2-specific)-Fc fusion proteins (data not shown).

If an Fc-antibody fragment is fused to the N-terminus of an scTNF-SFfusion protein (cf. FIG. 10), the amino acid sequence of the Fc-moduleis preferably as shown in SEQ ID NO: 38:

  1 METDTLLLWV LLLWVPAGNG DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT 61 CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK121 CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE181 WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS241 LSLSPG

Amino acids 1-20 (underlined) constitute an N-terminal secretory signalpeptide.

For connecting the Fc-module to the ScTNF-SF fusion protein, preferablyGly/Ser linkers are used. All linkers preferably start with a serine andpreferably end with glycine or serine. Preferred linker sequences 11-12are shown in the following:

11. (S)_(a)(GS)_(b)G(S)_(c) (a, b = 0, 1-6;  c = 0 oder 1) 12.S(GGGS)_(a)G_(b)(S)_(c) (a, b = 0 ,1-6;  c = 0 oder 1)

7.3 Dimerisation of the Single-Chain Fusion Proteins of the Invention7.3.1 Single-Chain Fusion Polypeptides Comprising One Additional Domain

The trimeric fusion proteins of the invention can further be dimerised.

In one embodiment, dimerisation will be obtained if the C-terminus of afirst fusion protein is directly connected to the N-terminus of a secondfusion protein via a linker structure as defined herein (FIG. 12).

In another embodiment, a fusion protein of the invention comprising anFab-antibody fragment as an additional domain, may be connected via alinker as defined herein directly with a further fusion protein of theinvention or indirectly via an scFv-antibody fragment fused to a furtherfusion protein of the invention (FIG. 13). Thereby, dimerisation of thetrimeric fusion proteins of the invention is accomplished.

In another embodiment, dimerisation of trimers may be obtained via theassembly of two fusion proteins of the invention comprising aFab-antibody fragment as an additional domain (FIG. 14). In this case,intermolecular disulfide bridges are formed.

For the construction of dimerising Fab fragments N-terminal to thescTNF-SF domain (e.g. FIG. 14), preferably the natural cysteine residuesof the IgG hinge region (SEQ ID NO: 35) are used.

Preferably the C-terminal cysteine of the Fab-sequence corresponds tothe C1-residue of the hinge region, which forms a disulfide bond withthe light chain. The second cysteine C2 may be used for the covalentlinkage of two Fab-modules. A third cysteine residue C3 may be open orlinked with the C3 of the neighbouring chain. Preferred linkers betweenthe Fab heavy chain sequence and the N-terminus of the scTNF-SF domainare linkers 13-22 as shown below.

13. DKTHT

PGSS(GS)_(a)G(S)_(b) 14. DKTHT

PGSS_(a)G(S)_(b) 15. DKTHT

(GSSGS)_(a)GSG(S)_(b) 16. DKTHT

GSS(GS)_(a)G(S)_(b) 17. DKTHT

GSS_(a)G(S)_(b) 18. DKTHT

(GSSGS)_(a)GS(G)_(b) 19. DKTHT

PP

PGSSGSGSGS(G)_(b) 20. DKTHT

PP

P(GSSGS)_(a)GS(G)_(b) 21. DKTHT

PP

PGSS(GS)_(a)GS(G)_(b) 22. DKTHT

PP

PGSS_(a)GS(G)_(b)

Further, the linkers may be modified by incorporation of N-glycosylationmotifs as described above.

In a further embodiment, dimerisation of the fusion proteins of theinvention comprising an Fc-antibody fragment as an additional N- and/orC-terminal domain, may be obtained by the formation of intermoleculardisulfide bridges between two of said fusion proteins. In that case,only one Fc-antibody fragment is present per dimer of a trimeric fusionprotein. Thereby, in contrast to the Fc-antibody fragment fusionproteins of the art, formation of higher molecular weight aggregates isnot very likely.

7.3.2 Single-Chain Fusion Polypeptides Comprising a Plurality ofAdditional Domains

The single-chain fusion polypeptide may comprise one or more additionaldomains, e.g. a further antibody fragment and/or a further targetingdomain and/or a further cytokine domain.

A fusion protein of the invention comprising an Fc-antibody fragment asone additional domain may be connected to a further Fab- orscFv-antibody fragment via the N-terminus of an N-terminal fusedFc-antibody fragment (FIG. 15) or directly via its N-terminus through afurther linker structure (FIG. 16), if the Fc-antibody fragment isconnected to the fusion protein of the invention via its C-terminus.

In addition to a further antibody fragment or instead of the furtherantibody fragment, a further cytokine, preferably an interleukin, may beconnected to the fusion protein. Thereby, it is possible to obtain acombination of an agonistic scCD95L and an antagonistic scCD95L moleculeor alternatively combinations of scTRAIL (R1-specific) and scTRAIL(R2-specific).

Said fusion proteins are especially useful for the induction ofapoptosis.

1. A single-chain fusion polypeptide comprising: (i) a first soluble TNFsuperfamily cytokine domain, (ii) a first peptide linker, (iii) a secondsoluble TNF superfamily cytokine domain, (iv) a second peptide linker,and (v) a third soluble TNF superfamily cytokine domain, wherein thefusion polypeptide is substantially non-aggregating.
 2. The polypeptideof claim 1, wherein the soluble TNF superfamily cytokine domain isselected from soluble CD95L or TRAIL domains.
 3. The polypeptide ofclaim 1, wherein the second and/or third soluble TNF superfamilycytokine domain is an N-terminally shortened domain which optionallycomprises amino acid sequence mutations.
 4. The polypeptide of claim 1,wherein at least one of the soluble TNF superfamily cytokine domains isa soluble CD95L domain with an N-terminal sequence which starts betweenamino acid Arg 144 and Val146 of human CD95L, and Arg144 and/or Lys145is optionally replaced by a neutral amino acid.
 5. The polypeptide ofclaim 4, wherein at least one of the soluble TNF superfamily cytokinedomains is a soluble CD95L domain with an N-terminal sequence selectedfrom (a) Arg144-(Gly/Ser) 145-Val (146) (b) (Gly/Ser) 144-Lys 145-Val(146) and (c) (Gly/Ser) 144-(Gly/Ser) 145-Val (146).
 6. The polypeptideof claim 2 wherein the soluble CD95L domain ends with Leu281 of humanCD95L and/or optionally comprises a mutation at position Lys177, and/orat position Tyr218.
 7. The polypeptide of claim 1 wherein at least oneof the soluble TNF superfamily cytokine domains is a soluble TRAILdomain with an N-terminal sequence which starts between amino acidGln120 and Val122 of human TRAIL and Arg121 is optionally replaced by aneutral amino acid, e.g. Ser or Gly.
 8. The polypeptide of claim 7,wherein at least one of the soluble TNF superfamily cytokine domain is asoluble TRAIL domain with an N-terminal sequence selected from (a)Arg121-Val122-Ala123 and (b) (Gly/Ser) 121-Val122-Ala123.
 9. Thepolypeptide of claim 7 wherein the soluble TRAIL domain ends with aminoacid G1y281 of human TRAIL and/or optionally comprises one or moremutations at positions R130, G160, H168, R170, H177, Y189, R191, Q193,E195, N199, K201, Y213, T214, S215, H264, I266, D267 or D269.
 10. Thepolypeptide of claim 1 wherein the first and second peptide linkersindependently have a length of 3-8 amino acids.
 11. The polypeptide ofclaim 1, which additionally comprises an N-terminal signal peptidedomain.
 12. The polypeptide of claim 1, which additionally comprises afurther domain in the N-terminal and/or C-terminal.
 13. A nucleic acidmolecule encoding the fusion polypeptide of claim
 1. 14. A cell or anon-human organism transformed or transfected with the nucleic acidmolecule of claim
 13. 15. A pharmaceutical composition comprising thefusion polypeptide of claim 1 and a pharmaceutically acceptable carrier,diluent and/or adjuvant.
 16. The polypeptide of claim 4, wherein thesoluble TNF superfamily cytokine domain is soluble TNF superfamilycytokine domain (iii) or (iv).
 17. The polypeptide of claim 6, whereinLys177 mutates to Glu, Asp or Ser, and Tyr218 mutates to Arg, Lys, Ser,or Asp.
 18. The polypeptide of claim 10, wherein the first and secondpeptide linkers are glycine/serine linkers.
 19. The polypeptide of claim11, wherein the N-terminal signal peptide domain comprises a proteasecleavage site.
 20. The polypeptide of claim 12, wherein the furtherdomain is a Fab or Fc fragment domain.